A. A. Klebanov

Testing of geroprotectors in experiments on cell cultures: Choosing the correct model system

We believe that cytogerontological models, such as the Hayflick model, though very useful for experimental gerontology, are based only on certain correlations and do not directly apply to the gist of the aging process. Thus, the Hayflick limit concept cannot explain why we age, whereas our “stationary phase aging” model appears to be a “gist model,” since it is based on the hypothesis that the main cause of both various “age-related” changes in stationary cell cultures and similar changes in the cells of aging multicellular organism is the restriction of cell proliferation. The model is applicable to experiments on a wide variety of cultured cells, including normal and transformed animal and human cells, plant cells, bacteria, yeasts, mycoplasmas, etc. The results of relevant studies show that cells in this model die out in accordance with the Gompertz law, which describes exponential increase of the death probability with time. Therefore, the “stationary phase aging” model may prove effective in testing of various geroprotectors (anti-aging factors) and geropromoters (pro-aging factors) in cytogerontological experiments. It should be emphasized, however, that even the results of such experiments do not always agree with the data obtained in vivo and therefore cannot be regarded as final but should be verified in studies on laboratory animals and in clinical trials (provided this complies with ethical principles of human subject research).

Culture medium pH and stationary phase/chronological aging of different cells

There is an opinion that the chronological aging (ChA) of yeast and the stationary phase aging (SPA) of cultured animal and human cells are a consequence of growth medium acidification. However, a number of recent publications indicate that, although this process has a certain influence on the rate of “aging” of cells in the stationary growth phase, it does not determine it completely. Apparently, the key factor in this case is the restriction of cell proliferation, which leads to cell “aging” even under physiologically optimal conditions. During yeast ChA and mammalian cell SPA, the medium is getting acidified to pH ≤ 4. Prevention of acidification can prolong the culture life span, but the cells will still die, although at a slower rate. Effects of medium acidification during ChA and SPA can be explained by activation of highly conserved growth signaling pathways leading to oxidative stress, and these processes, in turn, can play a role in aging of multicellular organisms and development of age-related diseases. Our previous experiments on the effect of buffer capacity of growth medium on SPA of transformed Chinese hamster cells showed that 20 mM HEPES had no effect on cell growth rate; in addition, the growth curves of experimental and control cells reached a plateau on the same day. However, the cell saturation density in the medium with HEPES was lower (i.e., the cells were “older” in terms of the gerontological cell kinetics model); on the other hand, the rate of SPA was markedly reduced, compared to the control, although the cells were still “getting older.” It can be assumed that extracellular pH (by the way, well correlated with intracellular pH) is an important factor (I.A. Arshavsky’s concept of the role of acidic alteration in aging) but not the key factor determining the survival of cells in a stationary culture.

Interpretation of data about the impact of biologically active compounds on viability of cultured cells of various origin from a gerontological point of view

Problems related to the interpretation of data obtained during testing of potential geroprotectors in cytogerontological experiments are considered. It is emphasized that such compounds/physical factors should influence the processes leading to the age-related increase of death probability of multicellular organisms (primarily human, in whose aging gerontologists are mainly interested). However, in the authors’ opinion, compounds that can be used to treat age-related diseases can hardly be classified as geroprotectors. It is noted that, in the model systems using cultured cells, researchers usually evaluate their viability, the criteria of which strongly depend on the aging theory that is shared by the experimenters. In addition, it is very important what cells are used in the studies—normal or transformed cells of multicellular organisms, unicellular eukaryotic or prokaryotic organisms, etc. In particular, the biologically active compounds that decrease the viability of cultured cancer cells, similarly to the compounds that increase the viability of normal cultured cells, may increase the life span of experimental animals and humans. Various problems with interpretation of data obtained with the Hayflick model, the stationary phase aging model, and the cell kinetics model, as well as in experiments on evaluation of cell colony-forming efficiency, are analyzed. The approaches discussed are illustrated on the example of the results of gerontological studies of rapamycin, a well-known mTOR inhibitor. It is assumed that factors retarding the stationary phase aging (chronological aging) of cultured cells are, apparently, the most promising geroprotectors, although the specific mechanisms of their action may vary considerably.

Some remarks on the relationship between autophagy, cell aging, and cell proliferation restriction

In the review, the main types of autophagy (macroautophagy, microautophagy, and chaperonemediated autophagy) are shortly described. Data about the character of the influence of autophagy on the aging process and on the development of some neurodegenerative diseases in various organisms are analyzed. It is noted that this effect is usually (though not always) beneficial. Results of investigations of the phenomenon in experiments on mice, nematodes, fruit flies, bacteria, yeast, and cell cultures of higher organisms are considered. Obvious relationship between autophagy activation and cell proliferation restriction is emphasized. The latter, in our opinion, is the main cause of age-related accumulation of various defects (the most important of them is DNA damage) in cells and tissues, which leads to an increase in the death probability (i.e., to aging). It is concluded that studies of the role of autophagy in the aging process on the models of chronological aging in yeast or stationary phase aging of cell cultures could be considered as the most appropriate approach to the problem solution.

Does Aging Have a Purpose

Ideas of proponents and opponents of programmed aging concerning the expediency of this phenomenon for the evolution of living organisms are briefly considered. We think that evolution has no “gerontological” purpose, because the obligate restriction of cell proliferation during the development of multicellular organisms is a factor that “automatically” triggers aging due to the accumulation of various macromolecular lesions in cells as a result of the suppression, or even complete cessation of emergence of new, intact cells. This leads to the “dilution” of stochastic damage (the most important of which is DNA damage) at the level of the entire cellular population. Some additional arguments in favor of the inexpediency of aging for both species and individuals are also listed.

Senescence-associated β-galactosidase—A biomarker of aging, DNA damage, or cell proliferation restriction?

The most popular biomarker of cellular senescence (BCS) is the activity of senescence-associated β-galactosidase (SA-β-Gal). Today, this is the prevailing BCS in the studies based on the definition of cell senescence (which we do not accept) understood primarily as accumulation in the cells (most often—those not prone to replicative senescence) of certain BCS under the impact of various external factors causing DNA damage. However, some papers provide evidence that SA-β-Gal activity in the cells is not a good BCS, because it often depends not so much on age (in vitro or in vivo) as on the method of research, the presence of certain pathologies, and, what is most important, on the proliferative status of the cells studied. Apparently, the restriction of cell proliferation under certain conditions (due to differentiation, contact inhibition, DNA damage, some diseases, etc.) is itself the factor that stimulates SA-β-Gal expression. In other words, SA-β-Gal appears even in “young” cells if their proliferation is suppressed. Such data, in our opinion, are additional evidence for the validity of our concept of aging, which postulates the leading role of cell proliferation restriction in the age-related accumulation of various macromolecular defects (primarily DNA damage) in cells.

On Choosing Control Objects in Experimental Gerontological Research

Recently, a large number of papers have appeared that describe the successful use of various biologically active compounds (short peptides, mitochondrial antioxidants, antidiabetic biguanides, mimetics of dietary restriction, autophagy modulators, etc.) as geroprotectors. However, in our opinion, in most cases, the positive results of such studies are determined by a “successful” selection of control objects. Animals with certain abnormalities are often used for this purpose, so that any favorable effect on the corresponding pathological processes leads to an increase in their lifespan. In addition, control animals can be normal (i.e., wildtype) but placed under certain extreme conditions that can be overcome just by using certain biologically active compounds. Thus, in this case, the treatment of pathologies rather than the effect on fundamental processes of aging is observed. There is a point of view that the results of Clive McCay’s well-known experiments, which have significantly prolonged the life of rats by limiting caloric intake, were determined by the facts that, firstly, the control animals fed ad libitum (which is absolutely untypical for animals in the wild) and, secondly, Fisher-344 rats, which were used in these experiments, are short-lived. The above considerations, apparently, also apply to the gerontological experiments on cultured cells. In particular, we sometimes hear remarks from our colleagues regarding the model of “stationary phase aging” of cell cultures, which is used in our laboratory, due to the fact that most of the experiments are performed on transformed rather than normal cells. However, this approach seems to us quite justified, because the phenomenon of “stationary phase”/chronological aging is common to a wide variety of cells, including bacteria, yeasts, cyanobacteria, mycoplasmas, as well as animal and plant cells. Cells with an unlimited mitotic potential do not change either from experiment to experiment or during long-term cultivation both with and without subcultivation (within the framework of the stationary phase aging model), which cannot be said of the normal diploid fibroblasts, whose telomeres are shortened with each division. In the period from seeding to entering the stationary phase of growth, the cells divide up to ten times! We believe that, to search for effective geroprotectors that affect the fundamental mechanisms of aging, it is necessary to perform studies on “maximally healthy” animals or on “maximally stable” model systems.

Impairment of the Viability of Transformed Chinese Hamster Cells in a Nonsubcultured Culture under the Influence of Exogenous Oxidized Guanoside is Manifested Only in the Stationary Phase of Growth

Despite the fact that oxidation products of nucleotides and nucleosides are markers of oxidative stress, reports of the paradoxical ability of these compounds to protect cells from the harmful effects of reactive oxygen species began to appear more often. Among all nitrogenous bases, guanine is most susceptible to the influence of oxidative stress; therefore, guanosine is oxidized more often than other bases. In the present work, the effect of exogenous 8-oxo-2′-deoxyguanosine on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during cell proliferation slowing down within a single passage and subsequent “aging” in the stationary growth phase) of nonsubcultured transformed Chinese hamster cells was studied. We showed that the nucleoside is rapidly absorbed by the cells from the medium, but it does not affect the growth of the culture, and impairs the viability of the cells in the late stationary growth phase. Thus, no mitogenic or geroprotective effect of 8-oxo-2′-deoxyguanosine was found.